The progression of many life-threatening diseases such as cancer, AIDS, infectious diseases, immune disorders and cardiovascular disorders is influenced by multiple molecular mechanisms. Due to this complexity, achieving cures with a single agent has been met with limited success. Thus, combinations of agents have often been used to combat disease, particularly in the treatment of cancers. It appears that there is a strong correlation between the number of agents administered and cure rates for cancers such as acute lymphocytic leukemia. (Frei, et al., Clin. Cancer Res. (1998) 4:2027-2037). Clinical trials utilizing combinations of doxorubicin, cyclophosphamide, vincristine, methotrexate with leucovorin rescue and cytarabine (ACOMLA) or cyclophosphamide, doxorubicin, vincristine, prednisone and bleomycin (CHOP-b) have been successfully used to treat histiocytic lymphoma (Todd, et al., J. Clin. Oncol. (1984) 2:986-993).
The effects of combinations of drugs are enhanced when the ratio in which they are supplied provides a synergistic effect. Synergistic combinations of agents have also been shown to reduce toxicity due to lower dose requirements, to increase cancer cure rates (Barriere, et al., Pharmacotherapy (1992) 12:397-402; Schimpff, Support Care Cancer (1993) 1:5-18), and to reduce the spread of multi-resistant strains of microorganisms (Shlacs, et al., Clin. Infect. Dis. (1993) 17:S527-S536). By choosing agents with different mechanisms of action, multiple sites in biochemical pathways can be attacked thus resulting in synergy (Shah and Schwartz, Clin. Cancer Res. (2001) 7:2168-2181). Combinations such as L-canavanine and 5-fluorouracil (5-FU) have been reported to exhibit greater antineoplastic activity in rat colon tumor models than the combined effects of either drug alone (Swaffar, et al., Anti-Cancer Drugs (1995) 6:586-593). Cisplatin and etoposide display synergy in combating the growth of a human small-cell lung cancer cell line, SBC-3 (Kanzawa, et al., Int. J. Cancer (1997) 71(3):311-319).
Additional reports of synergistic effects are found for:                Vinblastine and recombinant interferon-β (Kuebler, et al., J. Interferon Res. (1990) 10:281-291);        Cisplatin and carboplatin (Kobayashi, et al., Nippon Chiryo Gakkai Shi (1990) 25:2684-2692);        Ethyl deshydroxy-sparsomycin and cisplatin or cytosine arabinoside (AraC) or methotrexate or 5-FU or vincristine (Hofs, et al., Anticancer Drugs (1994) 5:35-42);        All trans retinoic acid and butyric acid or tributyrin (Chen, et al., Chin. Med. Engl. (1999) 112:352-355); and        Cisplatin and paclitaxel (Engblom, et al., Br. J. Cancer (1999) 79:286-292).        
In the foregoing studies, the importance of the ratio of the components for synergy was recognized. For example, 5-fluorouracil and L-canavanine were found to be synergistic at a mole ratio of 1:1, but antagonistic at a ratio of 5:1; cisplatin and carboplatin showed a synergistic effect at an area under the curve (AUC) ratio of 13:1 but an antagonistic effect at 19:5.
Other drug combinations have been shown to display synergistic interactions although the dependency of the interaction on the combination ratio was not described. This list is quite extensive and is composed mainly of reports of in vitro cultures, although occasionally in vivo studies are included.
In addition to the multiplicity of reports, a number of combinations have been shown to be efficacious in the clinic. These are described in the table below.
DRUGREFERENCEDRUG 1DRUG 23Langer, et al. (1999)Cisplatin or+ UFT (Tegafur/Drugs 58 Suppl. 3:Vindesineuracil)71-75FDAa (Colon or RectalLeucovorin+ 5-FUCancer)FDA (Colon or RectalIrinotecan+ Leucovorin+ 5-FUCancer)FDA (Breast Cancer)Herceptin+ PaclitaxelFDA (Breast Cancer)Xeloda+ Docetaxel(other names:Capecitabine)FDA (Ovarian and LungPaclitaxel+ CisplatinCancer)FDA (Lung Cancer)Etoposide+ Other FDA-ap-proved Chemo-therapeutic agentsFDA (Lung Cancer)Gemcitabine+ CisplatinFDA (Prostate)Novantrone+ Corticosteroids(mitoxantronehydrochloride)FDA (Acute Nonlympho-Novantrone+ Other FDA-ap-cytic Leukemia)proved drugsFDA (Acute Nonlympho-Daunorubicin+ Other FDA-ap-cytic Leukemia/Acute(DNR,proved drugsLymphocytic Leukemia)Cerubidine)FDA (Chronic Myelo-Busulfex+ Cyclophospha-genous Leukemia)(Busulfan;mide (Cytoxan)1,4-butanediol,dimethanesulfon-ate; BU, Myleran)aFDA: United States Food and Drug Administration
In addition, certain other combinations can be postulated from various reports in the literature to have the potential for exhibiting non-antagonistic combination effects or clinical efficacy or accepted as the standard of care by region study groups. These are:
DISEASEDRUG 1DRUG 2DRUG 3(Colon Cancer)Oxaloplatin+ 5-FU (or FUDR)+ Leucovorin(Metastatic BreastTaxol+ DoxorubicinCancer)Adriamycin+ Cytoxan (cy-(doxorubicin)clophosphamide)Methotrexate+ 5-FU (or FUDR)+ CytoxanVinblastine+ Doxorubicin(Non-small CellCarboplatin+ TaxolLung Cancer)Cisplatin+ Docetaxel(Taxotere ®)Vinorelbine+ CisplatinIrinotecan+ Cisplatin(Small Cell LungCarboplatin+ TaxolCancer)Cisplatin+ Etoposide(Prostate Cancer)Estramustine+ TaxolEstramustine+ MitoxantroneEstramustine+ Taxotere(Hodgkin'sBleomycin+ VinblastineLymphoma)(as part ofABDV: Adriamy-cin, Bleomycin,DTIC, Vinblas-tine)(Non-Hodgkin'sCarboplatin+ EtoposideLymphoma)(as part of ICE:Ifosfamide,Carboplatin,Etoposide)(Melanoma)IL-2+ Cisplatin(Acute MyeloidDaunorubicin+ CytosineLeukemia)ArabinosideVincristine+ Doxorubicin(Bladder Cancer)Carboplatin+ TaxolCarboplatin+ GemcitabineGemcitabine+ TaxolVinblastine+ Doxorubicin(as part ofMVAC: Metho-trexate, Vinblas-tine, Adriamycin,Cisplatin)(Head and Neck5-FU (or FUDR)+ Cisplatin+ LeucovorinCancer)(Pancreatic Cancer)Gemcitabine+ 5-FU (or FUDR)AdditionalCombinations:Carboplatin+ 5-FU (or FUDR)Carboplatin+ IrinotecanIrinotecan+ 5-FU (or FUDR)Vinorelbine+ CarboplatinMethotrexate+ 5-FU (or FUDR)Idarubicin+ AraCAdriamycin+ VinorelbineSafingol+ Fenretinide
Despite the aforementioned advantages associated with the use of synergistic drug combinations, there are various drawbacks that limit their therapeutic use. For instance, synergy often depends on various factors such as the duration of drug exposure and the sequence of administration (Bonner and Kozelsky, Cancer Chemother. Pharmacol. (1990) 39:109-112). Studies using ethyl deshydroxy-sparsomycin in combination with cisplatin show that synergy is influenced by the combination ratios, the duration of treatment and the sequence of treatment (Hofs, et al., supra).
It is thus known that in order for synergy to be exhibited by a combination of agents, these agents must be present in amounts which represent defined ratios. Indeed, the same combination of drugs may be antagonistic at some ratios, synergistic at others, and additive at still others. It is desirable to avoid antagonistic effects, so that the drugs are at least additive. The present invention recognizes that the result obtained at an individual ratio is also dependent on concentration. Some ratios are antagonistic at one concentration and non-antagonistic at another. The invention ensures ratios of components in the synergistic or additive range by delivering these agents in formulations that maintain the desired or administered ratio when the target location in the subject are reached and by selecting the ratios to be predominantly non-antagonistic at a desired range of concentrations, since the concentration at the target may be different from that administered.
PCT publication WO 00/51641 describes administering a combination of antiviral agents which is said to be synergistic. In vitro tests were used to determine synergistic ratios. However, there is no teaching of any mode of administration which would maintain this ratio in vivo. Indeed, the publication states that the components may be administered sequentially or simultaneously.
PCT publication WO 01/15733 describes putatively synergistic compositions for treating autoimmune disease. Again, the method of formulation does not ensure maintenance of this ratio after delivery.
Daoud, et al., Cancer Chemother. Pharmacol. (1991) 28:370-376, describe synergistic cytotoxic actions of cisplatin and liposomal valinomycin on human ovarian carcinoma cells. This paper describes an in vitro assay in which cisplatin which is free and, valinomycin which is encapsulated in liposomes are used to treat cultures of CaOV-3, a human ovarian tumor-derived cell line. The authors determined the concentration ranges over which synergism and antagonism was exhibited. Liposome encapsulation was employed to solubilize the valinomycin. As the experiments are performed in vitro, in vivo delivery is irrelevant.
U.S. Pat. No. 6,214,821 issued 10 Apr. 2001 to Daoud, describes pharmaceutical compositions containing topoisomerase I inhibitors and a staurosporine. The claims appear to be based on the discovery that staurosporines have the ability to abrogate topoisomerase I inhibitor-induced S-phase arrest and to enhance its cytotoxicity to human breast cancer cells lacking normal p53 function. No particular pharmaceutical formulation is suggested.
U.S. Pat. No. 5,000,958 to Fountain, et al., describes mixtures of antimicrobial agents encapsulated in liposomes which are said to exert an enhanced therapeutic effect in vivo. Suitable ratios of antimicrobial agents are determined by a combination effect test which empirically tests for synergy in vitro. There is no discussion of assuring a synergistic ratio over a range of concentrations.
Schiffelers, et al., J. Pharmacol. Exp. Therapeutic (2001) 298:369-375, describes the in vivo synergistic interaction of liposome co-encapsulated gentamicin and ceftazidime. The desired ratios were determined using a similar combination effect test to that of Fountain (supra), but there is no discussion of determination of a ratio wherein synergism is maintained over a range of concentrations.
The present invention recognizes, first, that it is possible to maintain a determined synergistic or additive ratio of therapeutic agents by controlling the pharmacokinetics of the formulation in which they are administered, and second, that the non-antagonistic ratio must be exhibited over a range of concentrations, since the concentration of components in a drug cocktail which reaches the target tissue may not be the same as that which is administered. The problem of maintaining synergy or additivity is solved by the recognition that when therapeutic agents are encapsulated in (i.e., stably associated with) delivery vehicles, such as liposomes, the delivery vehicles determine the pharmacokinetics and thus agents which are encapsulated will behave in a similar manner, and by selecting ratios which are predominantly synergistic/additive over a range of concentrations.